Staphylokinase is a profibrinolytic protein secreted by certain strains of Staphylococcus aureus that forms a stoichiometric complex with human plasminogen and displays localized plasminogen activation activity in a fibrin specific manner [Lack, C. H. (1948) Staphylokinase: an activator of plasma protease. Nature 161; Collen, D., De Cock, F., Vanlinthout, L, Declerck, P. J., Lijnen, H. R. and Stasen, J. M. (1992) Comparative thrombolytic and immunogenic properties of staphylokinase and streptokinase. Fibrinolysis 6; 232-242; Collen D., Lijnen, H. R. (1993) On the future of thrombolytic therapy for acute myocardial infarction. Am J Cardiol. 72; 46-50]. This is due to its ability to bind plasmin at the clot surface with nearly 150-fold higher affinity than the circulating plasminogen [Sakharov, D. V., Lijnen, H. R. and Rijken, D. C. (1996) Interaction between plasmin(ogen) and fibrin. J. Biol. Chem. 271; 27912-27918] where staphylokinase:plaminogen complex is rapidly inhibited by the blood component alpha 2-antiplasmin. In a clot environment plasminogen is partially degraded which results in conformational changes whereby binding with staphylokinase becomes stronger, therefore, resulting in a highly localized plasminogen activation activity around thrombi [Collen, D., De Cock, F., Vanlinthout, I., Declerck, P. J., Lijnen, H. R. and Stasen, J. M. (1992) Comparative thrombolytic and immunogenic properties of staphylokinase and streptokinase. Fibrinolysis 6; 232-242; Collen D., Wen, H. R. (1993) On the future of thrombolytic therapy for acute myocardial infarction. Am J Cardiol. 72; 46-50]. Since staphylokinase has a weak affinity for circulating but a high affinity for fibrin-bound plasminogen [Sakharov, D. V., Lijnen, H. R. and Rijken, D. C. (1996) Interaction between plasmin(ogen) and fibrin. J. Biol. Chem. 271; 27912-27918] it offers an advantage as a potential clot-dissolving agent with greater fibrin-specificity, considerably reduced antigenicity, and an efficacy at least as good as t-PA in terms of arterial patency [Vanderschueren S, Stockx L, Wilms G, Lacroix H, Verhaeghe R, Vermylen J, Collen D. (1995) Thrombolytic therapy of peripheral arterial occlusion with recombinant Staphylokinase. Circulation 92; 2050-2057; Vanderschueren, S., Van Vlaenderen, I. and Collen, D. (1997) Intravenous thrombolysis with recombinant staphylokinase versus tissue type plasminogen activator in a rabbit embolic stroke model. Stroke 28; 1783-1788].
Staphylokinase is a single chain 16 kDa protein, consisting of 136 amino acid residues. It forms a bimolecular complex with the blood proteins, such as plasminogen (PG) and plasmin (Pm) and exerts its fibrinolytic effects through conversion of an active non-specific serine protease, plasmin (Pm) to a highly specific proteolytic enzyme that can recognize blood zymogen, PG, as a substrate and convert it into plasmin that is capable of degrading blood clots. In a plasma milieu, SAK is able to dissolve fibrin clots without any associated fibrinogen degradation [Lijnen H. R., Van Hoef B., De Cock F., Okada K., Ueshima S., Matsuo O., Collen D. (1991) On the mechanism of fibrin-specific plasminogen activation by staphylokinase. J Biol. Chem. 266; 11826-11832; Collen D., Lijnen, H. R. (1993) On the future of thrombolytic therapy for acute myocardial infarction, Am J Cardiol. 72; 46-50]. Clinical trials have shown that Staphylokinase is as effective as t-PA at achieving early perfusion in myocardial infarction patients and its utility in thrombolytic treatment has now been established by several limited clinical trials [Collen D., Lijnen, H. R. (1993) On the future of thrombolytic therapy for acute myocardial infarction. Am J Cardiol. 72; 46-50; Lijnen, H. R., Collen, D. (1996) Staphylokinase, a fibrin-specific bacterial plasminogen activator. Fibrinolysis, 10; 119-126].
Staphylokinase is produced in very low amounts by its natural host, Staphylococcus aureus [Lack, C. H. (1948) Staphylokinase: an activator of plasma protease. Nature 161]. Considering its therapeutic applicability and clinical implications in thrombolytic therapy, several alternative sources of SAK production have been developed through recombinant routes. The staphylokinase gene has been cloned from the bacteriophages sakC [Sako, T., Sawaki, S., Sakurai, T, Ito, S., Yoshizawa, Y., Kondo, I. (1983) Cloning and expression of the staphylokinase gene of Staphylococcus aureus in Escherichia coli. Mol. Gen. Genet. 190; 271-277) and sak42D (Schlott, B., Hartmann, M., Guhrs, K. H., Birch-Hirschfeild, E., Pohl, H. D., Vanderschueren, S., van de Werf, F., Michoel, A., Collen, D. and Behnke, D. (1994) High yield production and purification of recombinant staphylokinase for thrombolytic therapy, Biotechnology 12; 185-189] as well as from the genomic DNA of a lysogenic Staphylococcus aureus strain [Behnke, D., Gerlach, D, (1984) Cloning and expression in Escherichia coli, Bacillus subtilis and Streptococcus sanguis of a gene for staphylokinase, a bacterial plasminogen activator. Mol. Gen. Genet. 210; 528-534]. The staphylokinase gene encodes a protein of 163 amino acids, with amino acid 28 corresponding to the NH2-terminal residue of full-length mature staphylokinase. The gene encoding for SAK has been overexpressed into various heterologous hosts, e.g., E. coli, Bacillus and Yeast [Sako, T., Sawaki, S., Sakurai, T, Ito, S., Yoshizawa, Y., Kondo, L (1983) Cloning and expression of the staphylokinase gene of Staphylococcus aureus in Escherichia coli. Mol, Gen. Genet. 190; 271-277; Behnke, D., Gerlach, D. (1984) Cloning and expression in Escherichia coli, Bacillus subtilis and Streptococcus sanguis of a gene for staphylokinase, a bacterial plasminogen activator. Mol. Gen. Genet. 210; 528-534; Schlott, B., Hartmann, M., Guhrs, K. H., Birch-Hirschfeild, E., Pohl, H. D., Vanderschueren, S., van de Werf, F., Michoel, A., Collen, D. and Behnke, D. (1994) High yield production and purification of recombinant staphylokinase for thrombolytic therapy. Biotechnology 12; 185-189] to produce SAK in large quantity in purified form for testing its clinical applicability.
Currently, attempts are being made to commercialize Staphylokinase for clinical use after several successful clinical and animal trial studies [Vanderschueren, S., Barrios, L., Kerdsinchai, P., Van den Heuvel, P., Hermans, L., Vrolix, M., De Man F., Benit, E, Muyldermans, L., Collen, D., Van de Werf, F., (2001) A randomized trial of recombinant staphylokinase versus alteplase for coronary artery patency in acute myocardial infarction. Circulation 92; 2044-2049; Armstrong, P. W., Burton, J., Pakola, S., Molhoek, P. G., Betriu, A. Tendera, M., Bode, C., Adgey, A. A., Bar, F., Van de Well, F. (2003) Collaborative Angiographic Patency Trial of Recombinant Staphylokinase (CAPTORS II). Am Heart J 146; 484-488]. However, being a product of bacterial origin, Staphylokinase elicits considerable allergic response during drug administration [Collen, D., De Cock, F., Vanlinthout, I., Declerck, P. J., Lijnen, H. R. and Stasen, J. M. (1992) Comparative thrombolytic and immunogenic properties of staphylokinase and streptokinase. Fibrinolysis 6; 232-242]. Attempts have been made to reduce its anitigenicity through the development of various mutant forms of Staphylokinase [Collen, D. (1996) Fibrin-selective thrombolytic therapy for acute myocardial infarction. Circulation 93; 857-865] where distinct mutations were created within its antigenic epitopes. Another limiting factor of Staphylokinase, that can hamper its use in thrombolytic therapy, is its relatively short plasma half-life (3-4 min) due to that repeated dose of this drug might be required to get effective recanalization during thrombolytic therapy and that in turn might exert higher allergic response in the patients. Therefore, development of second-generation SAK derivatives, where these shortcomings of native SAK are eliminated, would prove more advantageous. To overcome these problems, derivatives of SAK carrying PEG attachment within the protein at various sites have been generated [Vanwetswinkel, S., Plaisance, S., Zhi-Yong, Vanlinthout, I., Brepoels, K., Lasters, I., Collen, D., and Jespers, L. (2000) Pharmacokinetic and thrombolytic properties of cysteine-linked polyethylene glycol derivatives of staphylokinase. Blood. 95; 936-942; Verhamme, P., Goossens, G., Maleux, G., Collen, D. and Stas, M. (2007) A dose-finding clinical trial of staphylokinase SY162 in patients with long-term venous access catheter thrombotic occlusion. J Thromb Thrombolysis. 24; 1-5], however, derivatives carrying PEG at internal sites displayed significantly lower specific activity [Vanwetswinkel, S., Plaisance, S., Zhi-Yong, Vanlinthout, I., Brepoels, K., Lasters, I., Collen, D., and Jespers, L. (2000) Pharmacokinetic and thrombolytic properties of cysteine-linked polyethylene glycol derivatives of staphylokinase. Blood, 95; 936-942] suggesting internal sites within the core region of SAK may not be suitable for the chemical modification of SAK.
Cysteine derivatives of Staphylokinase have been described in the prior art [Vanwetswinkel, S., Plaisance, S., Zhi-Yong, Vanlinthout, I., Brepoels, K., Lasters, I., Collen, D., and Jespers, L. (2000) Pharmacokinetic and thrombolytic properties of cysteine-linked polyethylene glycol derivatives of staphylokinase. Blood. 95; 936-942; U.S. Pat. No. 6,383,483 “Staphylokinase derivatives with cysteine substitutions”; U.S. Pat. No. 6,902,733 “Staphylokinase derivatives with polyethylene glycol”] where cysteine residue has been substituted within the core region and amino-terminal part of Staphylokinase. Derivatization of cysteine substituted SAK mutants with PEG within the core region resulted in substantial loss of its plasminogen activation ability. Therefore, ideal site for the PEG conjugation within the core region has not been found and the approach to conjugate PEG with SAK has not been successful as these SAK derivatives display significantly lower plasminogen activation ability than the native form of SAK. Recombinant Staphylokinase variants obtained by site-directed substitution with cysteine, within the NH2-terminal region of SAK (serine 2 [Ser2] and/or Ser3), that is released from the core of the protein during plasminogen activation process, were derivatized with thiol-specific (ortho-pyridyl-disulfide or maleimide) polyethylene glycol (PEG) molecules, resulting in a SAK derivative that displayed a plasma half-life 4-5 fold higher (˜13 min) than the unmodified form (>3 min). The specific activity and thrombolytic potency of this SAK derivative in human plasma was found comparable to that of native SAK and currently this SAK variant is under clinical trial [Verhamme, P., Goossens, G., Maleux, G., Collen, D. and Stas, M. (2007) A dose-finding clinical trial of Staphylokinase SY162 in patients with long-term venous access catheter thrombotic occlusion. J Thromb Thrombolysis. 24; 1-5]. Although circulating half-life of PEG-linked SAK derivatives, described in the known literature or disclosed in known patents [Johnson, C., Royal, M., Moreadith, R., Bedu-Addo, F., Advant, S., Wan, M., and Conn, G. (2003) Monitoring manufacturing process yields, purity and stability of structural variants of PEGylated staphylokinase mutant SY161 by quantitative reverse-phase chromatography. Biomed Chromatogr. 17; 335-344] have been claimed to increase to certain extent, their use as a single bolus injection for clinical intervention, might require further improvement in the stability and half life of SAK molecule. The engineering of SAK for further improvement has been limited due to its smaller size and difficulty in targeting specific regions of protein without compromising functional properties of SAK as most of the regions of either involved in the interaction with the partner plasmin(ogen) or substrate plasminogen [Parry, M. A., Fernandez-Catalan, C., Bergner, A., Huber, R., Hopfner, K. P., Scholott, B., Guhrs, K. H. Bode, W (1998). The ternary microplasmin-staphylokinase-microplasmin complex is a protease-cofactor-substrate complex in action. Nat. Struct. Biol. 10; 917-923].
The present invention, therefore, unravels a novel strategy for engineering a SAK molecule to improve its thrombolytic properties by enhancing its plasma half life and stability and a low immune reactivity. The said properties are achieved although their clot dissolving ability is maintained similar to that of the wild type molecule. Here, design and development of new SAK derivatives has been discussed where amino and/or carboxy terminal regions of SAK have been extended by introducing new amino acid sequences, particularly one or more cysteine residues, to create dimeric/multimeric forms of SAK, and their modification by attaching a PEG molecule of different sizes within the extended region so that the integrated PEG with SAK remains away from the core functional region and does not interfere with biological function of SAK but simultaneously can increase overall stability and shelf life of the protein. Moreover, the Staphylokinase derivatives, thus generated, can have extended in vivo plasma half-life, thus, creating new SAK mutants that can be more advantageous in thrombolytic therapy. In principal, the present invention, disclosed herein, relates to new derivatives of Staphylokinase displaying higher thermal stability and increased in-vivo half-life than the unmodified Staphylokinase.
Therefore, details disclosed in the present invention, provide new strategy and design for the modification of SAK for engineering and chemical modification of SAK for enhancing its thrombolytic potential. SAK derivatives, thus generated, having multimeric forms and/or conjugated with PEG, disclosed in the present invention, display significant improvement in their functional properties over an unmodified SAK form and other known derivatives with respect to stability and circulating half-life and can be more useful for clinical purposes for the treatment of cardiovascular complications providing the advantage of higher temperature stability that can increase the shelf life of the protein and extended half life that can reduce the requirement of repeated dose during thrombolytic therapy.
The prime objective of the present invention is to develop a cysteine variant of Staphylokinase wherein at least one cysteine residue is added at amino and carboxy-terminal extension of SEQ ID NO: 1.
Another object of the present invention is to develop biologically functional derivatives of SAK that can display higher thermal and protease stability so that the shelf life of protein is increased and its plasma half-life in vivo is extended so that it can be more beneficial for the thrombolytic therapy. Integration of these two attributes in a SAK molecule can tremendously increase therapeutic potential of Staphylokinase for the treatment of circulatory disorders.
The other objective of the present invention is to extend amino and/or carboxy terminal regions of SAK carrying one or more cysteine residues to alter subunit association properties of SAK and to conjugate different lengths of PEG molecule away from the main functional regions of protein so that biological activity of protein is not compromised after PEG attachment but simultaneously can provide protection to the molecule from the protease attack so that its circulating half life in vivo is extended.
Yet another objective of the invention is to prepare a piece of DNA carrying complete genetic information for the production of SAK derivatives in a suitable host such as E. coli, Bacillus, Yeast or any microbial system using known recombinant DNA techniques for high level intracellular production of various SAK derivatives so that large amount of SAK mutant proteins can be obtained in high yield.
Yet another objective of the invention is to prepare SAK derivatives in purified form using known protein purification techniques and then conjugate one or more PEG molecule within the extended region of SAK to prepare mono or di PEGylated forms of SAK.
Overall objective of the present invention, therefore, is to develop new derivatives of SAK that can display better stability, enhanced shelf life and extended plasma half-life in vivo. These attributes in the staphylokinase derivatives, disclosed herein, will significantly improve thrombolytic potential of staphylokinase for the treatment of cardiovascular disorders.